Establishing an in vitro approach to generate osteomorphs

Introduction: Osteoporosis is a common bone disease caused by bone loss due to increased osteoclast activity. Major causes include chronic inflammatory disorders, e.g. rheumatoid arthritis, estrogen deficiency, and systemic glucocorticoid treatment. Denosumab, an anti-RANKL antibody, efficiently blocks osteoclastogenesis, improves bone mineral density, and reduces fracture risk. However, its discontinuation has been associated with a rebound effect, leading to a rapid formation of osteoclasts and an increased fracture rate. The recently described “osteomorphs” have been suggested as potential mediators of this process. Apart from their challenging characterization, it is currently unknown how diseases or drugs affect osteomorph biology. Here, we aim to establish a cell culture model to study osteomorphogenesis in order to determine their regulation by disease modulators. Methods : Osteoclasts were generated from wild-type mice and reporter mice (mT/mG, Vav:cre-mT/mG). Various time points were selected to determine the dynamics of differentiation, fission and re-fusion of osteomorphs. Differentiated cells were identified as osteoclasts by positive TRAP staining of cells with at least three nuclei. Cellular identity was further validatedby qRT-PCR for specific marker genes such as Ctsk, Oscar, Nfatc1, Trap, Rank, cFms, Dc and Oc-stamp. Flow cytometry (FACS) analysis was performed to distinguish osteomorphs from monocytes, macrophages, osteoclast precursor cells, and lymphocytes. Re-fusion capacity of osteomorphs was tested using fluorophore-labeled cells, complemented by viability assays and microscopy. Results : RANKL exposure (50 ng/mL) for 5-6 days induced osteoclast formation through fusion events during differentiation visible with live cell imaging. Withdrawal of RANKL for 2 days led to marked changes in the osteoclast recycling process. Approximately 65% of supernatant-derived cells—classified as osteomorphs—remained viable. FACS identified these cells as CD45+ CD115− RANK+ CD11b+ Ly6c+. Gene expression analysis demonstrated a downregulation of several osteoclast-specific marker genes. Although the master transcription factor Nfatc1 was reduced, it remained detectable in osteomorphs. The cells continued to express Rank, while cFms expression was diminished, consistent with the FACS results.Supernatant-derived cells were replated and restimulated with RANKL for 3–4 days, inducing re-fusion and formation of TRAP-positive osteoclasts. Compared to initial osteoclasts, re-fused osteoclasts displayed an increased expression of marker genes and a significantly higher number of TRAP-positive cells. Furthermore, re-fused osteoclasts showed an increased total cell area and appeared double-positive under fluorescence microscopy, confirming the fusion of differentially labeled osteomorph populations. Discussion: Taken together, we established an in vitro protocol to simulate osteoclast-osteomorph-recycling events.